The long term objective of this research is to define the molecular mechanism(s) by which an altered sarcoplasmic reticulum (SR) ryanodine receptor/Ca2+ release channel (RYR) results in the pharmacogenetic syndrome malignant hyperthermia (MH). In this anesthetic reaction that occurs in susceptible individuals during surgery, certain anesthetic agents and depolarizing muscle relaxants trigger a life-threatening muscle rigidity and rise in body temperature. This project will test four hypotheses using a purebred strain of autosomal recessive MH susceptible (MHS) pigs that possess the Arg 615 Cys RYR mutation in the skeletal muscle RYR isoform (ryr1). The first hypothesis to be tested is that metabolic factors in muscle contribute to the pathogenesis of the MH response. This will be tested by determining the effect of inorganic phosphate and other metabolites whose concentrations change significantly during exhaustive, MH-like muscle contraction, anions that potentiate E-C coupling, as well as lipids and their metabolites, on the activity of the RYR in SR vesicles, planar lipid bilayers and skinned muscle fibers. The second hypothesis to be tested is that the interaction of MHS-triggering anesthetics with the RYR is modified by muscle metabolites and non-triggering anesthetics such as propofol. We will examine whether propofol, which neither triggers MH nor alters RYR activity, modifies the action of MH-triggering anesthetics such as halothane which significantly activate RYR activity. The third hypothesis to be tested is that the expression of the MHS ryr1 in the nervous system results in altered cell signalling in those neurons containing the MHS ryr1. In situ hybridization and immunocytochemistry will be used to localize the cell types expressing the different RYR isoforms. Primary cell cultures of neuronal cells will be developed to determine the effect of the MHS ryr1 allele on the regulation of cytoplasmic Ca2+ in these cells. The fourth hypothesis to be tested is that the proteins derived from the different RYR genes and alleles may associate to form functional heterotetramers. Immuno-affinity chromatography with antibodies specific for the different alleles of the pig ryr1 gene will be used to determine the subunit composition of the RYR in the muscles of heterozygous MHS animals. A similar approach will be used to examine the subunit composition of the RYR in regions of the nervous system that express multiple RYR isoforms. These results will be particularly important in MH, as the functional activity of the RYR channel would likely be very different depending on whether it is comprised of a mixture of homotetramers of the MHS or normal ryr1, or rather is associated in heterotetramers with the ryr2 or ryr3 isoforms. Further understanding of the molecular basis of Mh will be of great assistance in the future identification, management and treatment of MHS individuals; will provide basic information regarding the protein complexes that effect Ca2+ regulation in muscle and neurons; and will be important in the future elucidation of the etiology of other neuromuscular diseases in which excitation-contraction coupling or Ca2+ regulation is abnormal.